Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder seen in reproductive-aged women and is characterized by chronic anovulation, infertility, insulin resistance, and hyperandrogenism. Abnormal neuroendocrine function appears to be an important pathophysiological factor in PCOS. By simultaneously deleting the Pro-opiomelanocortin (POMC) neuron leptin and insulin receptors in a murine model (IR/LepRPOMC), we created female mice that develop metabolic and reproductive features resembling PCOS spontaneously, without exposure to exogenous hormones. Interestingly, murine males with these genetic alterations also show increased LH and androgen production and impaired fertility, implying a shared mechanism between the genders. The overall goal of this proposal is to determine the underlying hypothalamic processes involved in the development of metabolic and reproductive abnormalities associated with a PCOS. We hypothesize that deletion of insulin and leptin signaling in POMC neurons reduces POMC-GnRH neuronal connections and/or beta-endorphin production by POMC neurons, thereby releasing GnRH neurons from inhibition. Specific AIM 1) Determine whether the leptin and insulin sensing by POMC neurons is required for normal neuronal connections to GnRH neurons in the hypothalamus. Immunohistochemical and double immunofluorescent techniques will be employed to evaluate the co-localization of POMC neurons with GnRH neurons within the hypothalamus during postnatal development. These experiments will determine if leptin and insulin sensing by POMC neurons play a role in neuronal development of the key hypothalamic neurons responsible for metabolic and reproductive homeostasis. Specific AIM 2) Establish whether the absence of leptin and insulin signaling in POMC neurons alters opioid production by POMC neurons. Using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA), we will directly measure the levels of processing intermediates and cleavage products within the ARC and pituitary tissues. These experiments will establish whether insulin and leptin signaling in POMC neurons regulate the production of prohormone convertases and how that effects endogenous opioid production. Specific AIM 3) Determine whether IR/LepRPOMC mice exhibit impaired sex steroid negative feedback. We will examine the effects of reduced leptin and insulin signaling in POMC neurons on pre-pubertal and pubertal development. We will then castrate IR/LepRPOMC males and administer exogenous testosterone to evaluate their sensitivity to negative feedback. Collectively, these experiments will establish whether the absence of insulin and leptin signaling in POMC neurons elevates LH pulse secretion and testosterone release, thus impairing the steroidal negative feedback.